Generation of Sugct knockout mice
To establish the role of the C7orf10 gene in vivo in mammals, we generated Sugct knockout mice by inserting loxP sites flanking the third exon of the Sugct gene (Figure S1a, Table S1). The targeting vector was electroporated into embryonic stem cells (ESCs) to generate heterozygous Sugct+/flox ESCs via homologous recombination (Figure S1b). Sugct+/flox ESCs were then injected into mouse blastocysts and targeted mice were obtained by standard procedures . Sugctflox/flox were bred with β-actin-cre mice to generate heterozygous null (Sugct+/null) animals, which were then intercrossed to get homozygous null animals (Sugctnull/null; hereinafter referred to as SugctKO). PCR genotyping of Sugct+/flox and SugctKO mice revealed bands at the expected sizes (Figure S1c). Homozygous SugctKO mice were obtained at the typical Mendelian frequencies (Figure S1d) and were viable.
To investigate Sugct/SUGCT expression levels in various tissues and verify the efficiency of the constructed SugctKO, we isolated RNA and proteins from several WT and SugctKO organs. Unlike in the mutant animals, we detected the expression of Sugct/SUGCT in WT kidney and liver at the mRNA and protein levels, respectively (Figure S1e, f). Both tissues are known to contain a high number of mitochondria due to their elevated metabolic rate and are often challenged by toxins and pathogens. Interestingly, it has been shown using single-cell transcriptomics in mouse kidney that Sugct is mainly expressed in proximal tubules, which are immune responders to toxic injuries . Accumulation of urinary metabolites in kidney, where SUGCT is mostly expressed could cause deleterious phenotypes, and therefore, we started our study with investigations of renal mouse tissue.
Metabolic changes in SugctKO mouse kidney
Patients with GA3 disease are known to excrete high levels of glutarate with no significant changes of acetylcarnitine, 3-hydroxyglutarate, glutarylcarnitine, and glutarylglycine in the urine . To investigate whether these metabolites can be detected in SugctKO mice, we performed untargeted metabolomics using Liquid Chromatography–Mass Spectrometry (LC–MS) in extracts isolated from WT and SugctKO kidney. All experimental mice were co-housed and matched gender- (male), age- (15 weeks), and backgroundwise (C57BL/6J), which means that usually, 2 wild type and 2 SugctKO mice were housed in a single cage. We detected a total of 669 compounds and tentatively annotated metabolites with an associated unique molecular mass, which in numerous cases was followed up by identity confirmation either via chemical standards or using metabolite fragmentation against libraries for possible matches  (Tables S2, S3, see “Materials and methods”). The data were visualized in a Volcano plot representing the differences in detected metabolites between WT and SugctKO mouse kidney (Fig. 2a). Among all identified compounds, differences in 12 metabolites were considered significant (2.2% of all metabolites; q value of 0.15, 15% False Discovery Rate (FDR), Fc ≥ 1.2). Importantly, amidst shortlisted metabolites were those involved in tryptophan catabolism (Fig. 2b). In line with the urinary metabolic alterations characteristic for patients with GA3 , the 11-fold increase in glutarate levels was the most prominent in SugctKO mouse kidney (Fig. 2b, c). Besides glutarate upregulation, we did not detect significant changes in acetylcarnitine, 3-hydroxyglutarate, glutarylcarnitine, and glutarylglycine in SugctKO mouse kidney (Figure S2a, Table S2) consistent with the previous clinical reports for GA3 . Unfortunately, due to technical limitations, we were unable to detect lysine and its co-metabolites. Intriguingly though, tryptophan degradation via 5-hydroxy-l-tryptophan (Fig. 2b, c) was found downregulated despite unchanged tryptophan levels (Fig. 2b), which might suggest alternative degradation pathways for tryptophan. Thus, our results indicate that besides the reported altered levels of glutarate in urine of patients with GA3, there may be also changes in other metabolites.
To investigate whether we can identify other metabolites related to GA3 in SugctKO mice, we expanded the analysis of our metabolomic data. The vast majority of significantly altered kidney metabolites (80%) were upregulated in SugctKO mice with only few downregulated, suggesting that SUGCT is a “repressor” of those compounds (Fig. 2c, Table S2). To get a more expanded view of the metabolic changes, we lowered the FDR to 25%, which allowed us to detect besides tryptophan co-metabolites (5.5%), also differentially regulated acylcarnitines (25.5%) and lipids (20%) (Figures S2b, S2c, Table S2). Most of these metabolites were only modestly increased, but these data still give an impression of the direction of the metabolic rewiring in SugctKO kidney. 30% of detected lipids were prenols (arnamiol, armillarin, valtrate, isopetasoide, icariside B8), while the rest, where medium/long-chain fatty acids, one glycerophospholipid, and acyl choline (Table S2). Intriguingly, among the detected lipids were those associated with gut microflora metabolism, such as adipic acid , as well as those contributing to dysbiosis (palmitic acid, oleic acid, and linoleic acid) . In addition, 24% of detected metabolites either did not have a eukaryotic origin and/or are co-regulated by gut bacteria (Fig. 2c, S2c, S3, Table S2, S3), indicating a prokaryotic contribution to the results that we observed. Considering that the kidney is held in a sterile environment, any presence of the non-host-derived metabolites was surprising. Interestingly, it has been shown that a substantial amount of the dietary tryptophan in the human gut is metabolized by bacteria . In accordance with this, we detected in SugctKO mouse kidney increased indoleacrylic acid, which derives from indole-propionic acid , and is known as a suppressor of commensal inflammation . It is important to keep in mind that gut bacteria-derived metabolites in kidney are pathological and linked to an early decline in renal function [20, 21] (Figure S3).
16S rRNA microbiome sequencing from stool DNA
Following recent improved understanding of the relation between the gut microbiome and human health , we wanted to get an overview of the bacteria species in the gut of WT and SugctKO mice and performed 16S rRNA microbiome sequencing from stool DNA. We ensured exposure to the same microbial environment by co-housing both groups of mice and found substantial differences in the proportion and also type of bacteria species detected. In particular, there was a shift resulting in increase of firmicutes relative to bacteroidetes in SugctKO mice (Fig. 3a–c and Table S5). This is strongest in the Blautia genus containing the families Ruminococcaceae and Lachnospiraceae, which mirror the changes seen in a rat model of type 2 diabetes  as well as a metabolic syndrome in Mexican women with diabetes . The metabolic effect of these bacterial families has been attributed to presence of carbohydrate-active enzymes, sugar transport, and metabolic pathways in their genome . In addition, microbial diversity changes including the Blautia genus have also been linked to levels of indole-propionic acid  also identified in our metabolomics data. The second most increased genus, Adlercreutzia, has also been found increased in a diabetic rat model, and interestingly, this increase could be reverted by traditional Chinese medicine Xiexin Tang . The third most increased genus in SugctKO mice is Bilophila, which is known to promote inflammation and aggravates high fat diet-induced metabolic dysfunctions in mice . The fourth most increased genus AF12 has also been found increased in obese mice under high-fat diet . At the same time, the common health-promoting gut microbes from the Bifidobacterium genus  were decreased (Fig. 3a–c).
Our results indicate that loss of Sugct affects the microbiome with changes closely resembling observed microbiome disbalance in metabolic diseases like diabetes.
Antibiotic treatment reverses the metabolite profile of SugctKO mice
To investigate the potential contribution of intestinal bacteria to the metabolic changes detected in the SugctKO mouse kidney, we eliminated gut bacteria residing in the intestines by treatment with broad-spectrum antibiotics. The efficiency of antibiotic administration (hereinafter referred to as “abx”) on intestinal bacteria clearance was tested by 16S rRNA gene amplification of bacterial nucleic acids extracted from feces (Figure S4a, see also “Materials and methods”) . We were not able to detect bacterial DNA after antibiotic treatment, indicating that the number of bacteria in the intestines of our mice was below the detection limit.
To compare WT and SugctKO mice prior and post-antibiotic administration, we collected blood from the same animal and the plasma was then subjected to metabolomic analysis by untargeted LC–MS. Using the same annotation criteria as described for the kidney (see “Materials and methods”), among 416 detected peaks (Fig. 4a–d, Table S4), we found that 145 and 151 metabolites were significantly changed after antibiotic treatment in WT and SugctKO, respectively (Fig. 4c, d). Furthermore, we tentatively annotated 14 compounds with significantly altered levels in WT vs SugctKO (Fig. 4a, 14/416), which were reduced to 1 metabolite after antibiotic treatment (Fig. 4b, 1/416), indicating that the differences in metabolites between WT and SugctKO were mostly governed by gut microbiota. Both control and mutant animals treated with antibiotics displayed very low levels of bacterial metabolites, further confirming the efficiency of the gut microflora clearance (Fig. 4e, green bar). In agreement with the LC–MS study in kidney (see Fig. 2), the alterations of metabolite expression in SugctKO mouse plasma, when compared to WT, included mainly lipids and acylcarnitines (Fig. 4e). Among the upregulated lipids (32%) were mostly lysophospholipids and phosphatidylserines (Fig. 4e, Table S4), which are known to accumulate in organs with high metabolic activity, as liver  and brain , but not in plasma. High levels of lysophospholipids in the bloodstream are linked to renal failure in hemodialysis , while the presence of phosphatidylserines is still not understood. Despite increased lipid levels, acylcarnitines appeared to be downregulated after antibiotic treatment (Fig. 4e, Table S4), suggesting that they may originate either directly or indirectly from microbiota. We also found significantly decreased concentrations of glycine-conjugated compounds, which serve as phase II metabolic products in chemical detoxification processes and are associated with the action of gut microflora  (Fig. 4e, pink bar). As expected, upon antibiotic administration, the levels of glycine conjugates became similarly low in WT abx and SugctKO abx plasma (pink bar, Fig. 4e, Table S4). Of interest, among other significant metabolites detected only in SugctKO mouse plasma (Fig. 4e, black bar) were dipeptides (Table S4). Although the consequences of dipeptides in plasma remain elusive, it is known that the clearance of not fully digested proteins depends on kidney and intestine functions . In addition to metabolomic differences in SugctKO mouse plasma, we did not detect any morphological changes in kidney of young SugctKO mice prior and post abx treatment (Figure S4b). Nevertheless, we observed a mild increase of lipids in SugctKO mouse kidney that disappeared in gut microflora-deprived mice (Figure S4c, d), which agrees with the lipid imbalance in SugctKO mouse kidney (see Figs. 2c, 4e, S2, S3, Tables S2, S4).
Altogether, our data suggest that the antibiotic clearance of gut microflora in SugctKO mice alleviates alterations in the levels of lipids, acylcarnitines, bacterial metabolites, glycine conjugates, and dipeptides in SugctKO mouse plasma. This indicates that the absence of the gut microbiome restores the metabolic homeostasis in the animals harboring the Sugct mutation and may also suggest that the gut microbiome plays an important role in the GA3 disease.
Age-associated obesity and glucose intolerance in SugctKO mice
Initially, we did not observe obvious phenotypic changes in young SugctKO mice and their WT counterparts. However, renal lipidosis (see Fig. 2 and S2, S3, S4c, d) is associated with age-related progressive kidney failure and as a consequence, decreased physical and functional well-being of the patients [35, 36]. Based on this, we hypothesized that Sugct mutations could affect kidney functions in an age-dependent manner. Therefore, we aged WT and SugctKO animals for 52 weeks and monitored their body weight (Fig. 5a). Interestingly, the body weight of SugctKO mice was significantly elevated (≈ 41.5 g) when compared to their co-housed WT equivalents (≈ 36.7 g) at 52 weeks.
The significant increase of body weight in SugctKO mice could indicate metabolic dysfunction due to the loss of Sugct. Since SUGCT is highly expressed in kidney (see Figure S1e, f), where we observed metabolic changes in SugctKO mice (see Fig. 2, S2, S3), we collected kidneys from 52- to 58-week-old WT and mutant animals. We detected elevated number of vesicles in cytoplasm of renal tubular epithelial cells in WT mice , which in a subset of experimental SugctKO animals were further elevated (Fig. 5b, top panel). In addition, we noticed an increase of interstitial mononuclear cell infiltrate in SugctKO in comparison with WT mice (Fig. 5b, lower panel), an indication of inflammation. Therefore, we investigated the number of macrophages by tissue staining with F4/80 antibodies, best known as a marker of mature mouse macrophages and microglia  (Fig. 5c). We detected a threefold increased staining of macrophages in kidneys of aged SugctKO mice when compared to WT animals (Fig. 5d), which supports our hypothesis that metabolic changes in SugctKO mice may promote an inflammatory response.
Furthermore, there was lipid accumulation in the SugctKO kidney (Fig. 5e, f). Beside kidney, we analyzed histopathological changes in the liver (Figure S5a) and epididymal white adipose tissue (ewat; Fig. 5g, S5b) due to the observed weight gain in mutant animals (Fig. 5a). We observed micro- and macrovesicular steatosis in SugctKO mouse liver (Figure S5a) and we detected lipids by Oil Red O staining (Figure S5c, quantification shown in S5d). In addition, SugctKO mice displayed a greater degree of inflammation in adipose tissue, often forming “crown-like structures”  (Fig. 5g). This type of adipose tissue pathology indicates adipocyte death, which is often associated with macrophages surrounding dying adipocytes . Despite the histopathological changes detected in kidney, liver, and epididymal white adipose tissue (ewat), no additional gross abnormalities were found in aged mutant animals.
In summary, we uncovered that ageing significantly contributes to the phenotype in SugctKO mice through increased body fat accumulation and progressive renal tubular vacuolation, which was accompanied with increased macrophage levels, fat accumulation in liver, and adipocyte death.
Lysine-enriched diet aggravates histopathological changes in SugctKO mouse kidney
Diet composition has far-reaching effects on mammalian physiology . Certain diet-induced pathologies that are severe in humans might only appear later or never in mice, since they are not exposed to varied diets. As previously shown in a mouse model for GA1, GcdhKO mice despite accumulating glutaric and 3-hydroxyglutaric acid, develop only mild motor deficits, unlike humans . However, 4-week-old GcdhKO mice exposed to high-lysine diet display severe striatal degeneration typical of the human GA1 disease and 75% of the mice die within 3–12 days .
To accelerate and/or aggravate pathological changes observed in aged SugctKO mice (see Fig. 5b–f, S5), 8–12 week-old WT and SugctKO animals were fed with high-lysine diet for 20 weeks. The choice of diet was based on a previously reported study on GcdhKO mice fed with high-lysine or high-protein but not high-tryptophan diet  that substantially aggravated the phenotype. Although the histopathology of WT and SugctKO mice exposed to lysine diet (hereinafter referred to as “Lys”) revealed inflammation accompanied by steatosis in both WT Lys and SugctKO Lys mouse liver (data not shown), morphological changes in kidney were only detected in SugctKO Lys mice (Fig. 6a). SugctKO Lys developed acerbated interstitial mononuclear cell infiltrate, medullary tubule mineralization, tubular proteinosis, tubule dilation, and cystic change accompanied with increased renal tubular vacuolation compared to their corresponding controls (Fig. 6a). The presence of inflammatory cells in SugctKO Lys mouse kidney was confirmed by F4/80 staining (Fig. 6b, c). The number of detected macrophages in SugctKO on normal diet was threefold higher when compared to WT, while SugctKO Lys displayed almost a 2.5-fold increase in respect to SugctKO with no observed change in WT Lys mouse kidney (Fig. 6c). Moreover, the inflammatory state in kidney of SugctKO mice on normal diet was accompanied by an increase in lipid accumulation, which was aggravated by approximately threefold in SugctKO mouse kidney in relative comparison to WT and increased by additional twofold in SugctKO Lys (Fig. 6d, e). Interestingly, the lipid levels in WT Lys were similar to SugctKO on normal diet (Fig. 6d, e). Taken together, increased intake of dietary lysine in the context of Sugct deficiency elevates lipid accumulation and inflammatory cells in the kidney, suggesting that diet could also be a factor in the development of the GA3 disease.